For 30 years the canonical function for desmoplakin has been to link intermediate filaments to desmosomes. This function has been confirmed by traditional knockouts and tissue specific knockouts. However, recent evidence strongly suggests that desmoplakin may also have nonconical functions. An example is the recent discovery of DP in capillaries and lymphatic vessels. DP has also been linked to the cell adhesion molecule, PECAM, which has a role in adhesion and signal transduction. In fact the relationship between PECAM and DP led me to investigate if DP may have a role (direct or indirect) in cell signaling. To do so, we employed a high-throughput proteomics assay whereby Luciferase-tagged DP cDNA bait was co-expressed with over 500 Flag-tagged prey cDNAs, followed by immunoprecipitation with a flag antibody and detection using the luciferase assay. Using this high throughput assay, we found three guanine exchange factors (GEFs) and 1 GTPase activating protein (GAP) directly associated with DP. The focus of this proposal is to characterize the GEF/DP and GAP/DP interaction in differentiating keratinocytes using several different methods as well as determining downstream effects of disrupting the GEF/DP and GAP/DP interaction by looking at Rho. [unreadable] [unreadable] [unreadable]